2.5 | Ultrasound-induced cytosolic delivery of Saporin
Finally, we demonstrated a control over cells in an ultrasound-induced
manner using the current cytosolic protein delivery system. Saporin
(Sap) was used as a cargo protein. Sap is a plant-derived
ribosome-inactivating protein that leads to cell death through its
introduction into the cytosol[35,36]. Sap has no
free thiol group; therefore, the amine group of the lysine residues was
thiolated with 2-iminothiolane
hydrochloride[37,38]. Thiolated Sap was conjugated
with PCNDs via disulfide bond formation in the same manner as that used
for β-Gal. After intracellular accumulation of PCNDs carrying Sap, the
cells were exposed to ultrasound, followed by incubation for 2 days.
Alive cells were stained with calcein-AM, and all cells were
subsequently analyzed for the presence of green fluorescence of calcein
using flow cytometry. The cell viability drastically decreased to below
30% after the treatment with both Sap-carrying PCNDs and ultrasound,
while the treatment with Sap-carrying PCNDs without exposure to
ultrasound barely reduced the cell viability (Figure 4). Although the
cell viability decreased to 66% even in the control experiment wherein
PCNDs without Sap were used, there was a significant difference in the
cell viability between PCNDs with and without Sap after an exposure to
ultrasound. These results indicated that Sap was released into the
cytosol upon exposure to ultrasound, resulting in a significant
contribution to decreased cell viability.